Premium
Displaced Conformational Equilibrium in a Piezophilic Cytochrome P450
Author(s) -
Sineva Elena V,
Halpert James R,
Davydov Dmitri R
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.512.6
Subject(s) - hydrostatic pressure , chemistry , substrate (aquarium) , heme , enzyme , cytochrome p450 , conformational change , stereochemistry , biochemistry , biology , thermodynamics , ecology , physics
Cytochromes P450 from piezophilic bacteria living at extreme pressures provide a powerful tool for exploration of the substrate‐dependent conformational transitions in the enzyme, which involve large changes in hydration and are thus pressure‐sensitive. Cytochrome P450 of a deep‐sea bacteria Photobacterium profundum SS9 was cloned and expressed in E. coli . Purified P450‐SS9 was found to be predominantly in the high‐spin state. The enzyme was found to bind various substrates including fatty acids and their derivatives. The spin equilibrium could be displaced by substrate binding to either high‐spin (HS) or low‐spin (LS), depending on the structure. In contrast to all cytochromes P450 studied up to date, hydrostatic pressure failed to displace the spin equilibrium completely to the LS state in the substrate‐free P450‐SS9. Similar behavior is observed in HS‐complexes, whereas the spin equilibrium in the LS‐complexes becomes pressure‐sensitive. We also probed the accessibility of the heme pocket with H 2 O 2 –dependent heme depletion. The rate of this process, which was very slow in the substrate‐free enzyme and in HS‐complexes, is dramatically increased in LS‐complexes. These findings provide evidence of large‐scale changes in the openness and degree of hydration of the active site that are involved in the substrate‐dependent spin transitions in the enzyme. (Supported by NIH grant GM054995)